Visualisation of PCNA monoubiquitination in vivo by single pass spectral imaging FRET microscopy.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 20126408.
- Also identified by DOI 10.1371/journal.pone.0009008 and PMC identifier 2814862.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
Monoubiquitination of the DNA sliding clamp, PCNA, plays a central role in the control of damage bypass during replication. By combining a widely-spaced FRET donor/acceptor pair (CFP and mRFP) with spectral imaging, we have developed a simple method for the visualisation of PCNA monoubiquitination in both fixed and live cells with a single imaging pass. We validate the method with genetic controls in the avian cell line DT40 and use it to examine the intracellular dynamics of PCNA ubiquitination following subnuclear UV irradiation. This general approach is likely to be of utility for live imaging of post-translational modifications of a wide range of substrates in vivo.
Medical subject headings
- Fluorescence Resonance Energy Transfer
- Proliferating Cell Nuclear Antigen
- Ubiquitination